The present invention relates generally to analyses for enzymatic substances and, more specifically, to methods and materials for the quantitative detection of BB and MB isoenzymes of creatine kinase by radioimmunoassay techniques.
Enzymes are proteinaceous substances produced by living cells. They function to bring about or accelerate chemical reactions in an organism without themselves undergoing marked chemical alteration in the process. One such enzyme, called creatine kinase, catalyzes an energy transfer (transphosphorylation) reaction, i.e., creatine+adenosine triphosphate.revreaction.creatine phosphate+adenosine diphosphate.
Creatine kinase enzyme is a dimeric molecule which exists in at least three combinative forms (isoenzymes), designated "MM," "MB" and "BB" on the basis of monomer combination. The M and B monomers which make up the enzymes each have a sulfydryl (SH) group-containing "active" site, the integrity of which is essential to catalytic activity. This active site is easily susceptible to inactivation upon contact with oxidizing materials. Each isoenzyme has a mass of approximately 82,000 daltons.
Analyses of profiles of creatine kinase isoenzyme (hereafter, "CK") activities in human tissue indicate that brain tissue ordinarily contains only the BB isoenzyme; skeletal muscle tissue ordinarily only the MM isoenzyme; and heart muscle tissue ordinarily both the MM and MB isoenzymes. Human plasma from normal subjects includes primarily the MM isoenzyme with less than 0.005 I.U. of MB CK per milliliter and no detectable quantities of BB CK.
After cerebral infarction, infection or other forms of cerebral damage, the concentration of MM isoenzyme in plasma is often somewhat elevated but the BB isoenzyme in plasma is generally not detectable. The lack of detectable BB CK is probably related to numerous factors including an effective blood/brain barrier. Damage to skeletal muscle by trauma or muscle disease such as muscular dystrophy is almost invariably accompanied by elevation of concentrations of the MM isoenzyme in plasma. Some very small increases in plasma BB activity have been reported in diseased muscle patients and immature muscle fibers of regenerating muscle have been proposed as the source of the BB in the blood.* Damage to heart muscle ordinarily results in elevation of plasma activity of both MM and MB CK. FNT *It has been reported that developmentally mature skeletal muscle may contain substantial quantities of BB CK which appears to be immunoreactive with a BB immune plasma but inactive when analyzed by cellulose acetate electrophoresis. [See, Armstrong, et al., J.Biol.Chem., Vol. 252, No. 10, 3105, 3112 and 3117 (1977).
Because the only human tissue containing appreciable amounts of MB CK is the myocardium, an elevation of MB concentration in blood--to the extent that it can be distinguished from increases in MM CK provides a remarkably sensitive and specific indicator of myocardial injury. Accordingly, analysis of serial changes in plasma MB CK has heretofore been employed to estimate the extent of acute myocardial infarction in experimental anaimals and human patients.
Analysis of a plasma sample for MB CK activity is frequently "indirectly" secured through prior art assays for enzyme activity. Employing a spectrophotometric technique for detecting NADPH developed from creatine kinase-catalyzed formation of ATP through coupled enzyme systems, relative increases or decreases in blood sample CK activity are predicated upon corresponding changes in catalytic activity of the enzyme isolated from the sample. The results of such prior art assays are directly dependent on several perameters, one of which is the rate of disappearance of the isoenzyme activity from plasma. Unfortunately, the factors responsible for disappearance of MB activity from the circulation have not been well elucidated. It is unclear, for example, whether disappearance of enzyme activity is ratelimited by inactivation, denaturation, or some form of removal of intact enzyme molecules from circulation. Consequently (and apart from the difficulty in distinguishing activity of MM CK from MB CK) activity-based determinations are subject to substantial unreliability.
The prior art provides certain proposals for radioimmunoassay ("RIA") analysis of plasma samples for use in quantitative detection of enzymes, and specifically creatine kinase isoenzymes, independently of analysis for enzyme activity. See, generally, the review article, "The Measurement of Enzymes by Radioimmunoassay" by J. Landon, et al. [Ann. Clin. Biochem., 14, pp. 90-99 (1977)] which recounts the relative superiority of RIA techniques to those based on catalytic activity. Simply put, according to one such RIA technique a stoichiometric excess of a pure, labelled (radioisotopic) material is allowed to associate (e.g., by antigen/antibody reaction) with the selected reactive substance such as antibody previously exposed to a sample containing an "unknown" quantity of enzyme which is unlabelled, but which is capable of a similar association. Direct quantitative information concerning the "unknown" concentration, as opposed to activity, is obtained on the basis of a count of radioactivity of the remaining selected substance which associates with the labelled material.
Nicholson et al., [Proc. Austral. Assoc. Neurologists, Vol. 10, pp. 105-108 (1973)] described a method involving labelling skeletal muscle MM creatine kinase with .sup.125 I and report development of an RIA for the MM isoenzyme which assertedly measures "enzymes independently of the integrity of the active site." The method employed by Nicholson, et al. for labelling human creatine kinase is that described by Hunter, et al. [Nature, Vol. 194, pp. 4956 (1962)], employing chloramine-T to directly introduce the desired isotope into tyrosyl and histidyl residues of the enzyme protein chain. This method, however, has been associated with considerable structural changes (i.e., tertiary structure destruction) and loss of MM isoenzyme activity through disruption of the sulfhydryl-group-containing active site. Because the assay is assertedly specific for the M subunit, it invites cross-reaction with MM CK and MB CK and is thus incapable of distinguishing elevation of serum MB concentration resulting from myocardial infarction from elevation of serum MM concentration resulting from skeletal muscle damage or disease. The chloramine-T reagent used in the labelling procedure of Nicholson, et al. cannot be used for labelling the BB or MB isoenzymes due to their relative lability (instability, visa-vis MM) in the presence of the highly oxidative --sulfhydryl-group-disrupting--chloramine-T reagent. In sum, the Nicholson, et al. proposal has have not provided a useful basis to develop an RIA specific for BB and MB isoenzymes.
A prior art publication of interest to the background of the invention is Fang, et al. [Biochem. Biophys. Res. Comm., Vol. 65, pp. 413-419 (1975)] which reports that creatine kinase enzyme activity losses from direct iodination by prior art chloramine-T, thallic trichloride and lactoperoxidase methods may be avoided through use, for iodination, of a Bolton-Hunter acylation reagent which conjugates (combines at a free amino (NH.sub.2) group of the protein, thus avoiding disruption of the active site. The reagent involved was an iodinated compound derived from N-succinimidyl-3-(4-hydroxyphenyl propionate). Fang, et al. labelled rabbit skeletal muscle MM CK. There is no mention of human MM CK nor was there any mention of animal or human MB or BB CK isoenzymes. While the reported preservation of enzyme active sites would have suggested that a more accurate RIA for MM creatine kinase than that of Nicholson, et al. might be secured, the prior art was still without a method for labelling the more labile MB and BB CK isoenzymes. Furthermore, the antibody noted in Fang, et al. was to rabbit MM CK which offered no specificity for human BB or MB CK isoenzymes. The necessary ingredients for a human CK isoenzyme RIA with a specific antibody to BB and MB CK and the necessary stabilizing conditions for such analysis were yet to be developed.